Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Substrate stiffness modulates human gingival fibroblast paracrine signaling to promote osteogenic differentiation of human periodontal ligament cells

doi: 10.3389/fbioe.2026.1753774

Figure Lengend Snippet: Substrate stiffness regulates the behaviors of human gingival fibroblasts (HGFs). Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect gene expression levels of (A) anti-inflammatory markers, IL4 , and IL10 , (B) matrix metalloproteinase markers, including MMP9 , and TIMP1 , (C) chemokine, CXCL12 . The expression of GAPDH was used as an internal control. Data were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests ( n = 3: P < 0.05). (D) The expression of GAPDH was used as an internal control. Data were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests ( n = 3: P < 0.05). (D) The protein expression of CXCL12 was detected by ELISA analysis. Data were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests ( n = 4: P < 0.05). Data are presented as the mean ± standard deviation (SD), with different letters indicating statistically significant differences between multiple groups. IL4, interleukin 4; IL10, interleukin 10; MMP9, matrix metalloproteinase 9; TIMP1, tissue inhibitor of matrix metalloproteinases 1; CXCL12, CXC motif chemokine 12; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDMS, polydimethylsiloxane.

Article Snippet: CXCL12 protein in conditioned medium was measured by Human CXCL12/SDF-1α ELISA Kit (Quantikine, R&D systems.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Control, Comparison, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Substrate stiffness modulates human gingival fibroblast paracrine signaling to promote osteogenic differentiation of human periodontal ligament cells

doi: 10.3389/fbioe.2026.1753774

Figure Lengend Snippet: Substrate stiffness regulates human gingival fibroblasts (HGFs) behaviors under an inflammatory condition. Real-time RT-PCR was performed to detect gene expression levels of (A) IL4 and IL10 , (B) MMP9 and TIMP1 , and (C) CXCL12 . The expression of GAPDH was used as an internal control. Data were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests ( n = 3: P < 0.05). (D) The expression protein of CXCL12 was detected by ELISA analysis. Data were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests ( n = 4: P < 0.05). Data are presented as the mean ± standard deviation (SD), with different letters indicating statistically significant differences between multiple groups. IL4, interleukin 4; IL10, interleukin 10; MMP9, matrix metalloproteinase 9; TIMP1, tissue inhibitor of matrix metalloproteinases 1; CXCL12, CXC motif chemokine 12; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDMS, polydimethylsiloxane; LPS, lipopolysaccharide.

Article Snippet: CXCL12 protein in conditioned medium was measured by Human CXCL12/SDF-1α ELISA Kit (Quantikine, R&D systems.

Techniques: Quantitative RT-PCR, Gene Expression, Expressing, Control, Comparison, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Substrate stiffness modulates human gingival fibroblast paracrine signaling to promote osteogenic differentiation of human periodontal ligament cells

doi: 10.3389/fbioe.2026.1753774

Figure Lengend Snippet: Mitogen-activated protein kinase (MAPK) pathway regulated substrate stiffness-induced CXCL12 expression in human gingival fibroblasts (HGFs). (A) Real-time RT-PCR was performed to detect gene expression levels of CXCL12 . The expression of GAPDH was used as an internal control. Data were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests ( n = 3: P < 0.05). (B) The protein expression of CXCL12 was detected by ELISA analysis. Data were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. ( n = 4: P < 0.05). Data are presented as the mean ± standard deviation (SD), with different letters indicating statistically significant differences between multiple groups. CXCL12, CXC motif chemokine 12; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDMS, polydimethylsiloxane.

Article Snippet: CXCL12 protein in conditioned medium was measured by Human CXCL12/SDF-1α ELISA Kit (Quantikine, R&D systems.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Control, Comparison, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Substrate stiffness modulates human gingival fibroblast paracrine signaling to promote osteogenic differentiation of human periodontal ligament cells

doi: 10.3389/fbioe.2026.1753774

Figure Lengend Snippet: Effects of conditioned media of human gingival fibroblasts (HGF-CM) under different substrate stiffness on human periodontal ligament cells’ behaviors. (A) Human periodontal ligament cells (HPDLCs) cultured with HGF-CM in osteogenic medium for 24 h. HPDLCs morphology was demonstrated using a phase-contrast microscope. Scale bars: 300 μm. (B) Real-time RT-PCR was performed to detect gene expression levels of CXCR4 , which is receptor of CXCL12. (C) Real-time RT-PCR was performed to detect gene expression levels of pro-inflammatory cytokine, IL1b . (D) Real-time RT-PCR was performed to detect gene expression levels of MMP8 and TIMP1 . The expression of GAPDH was used as an internal control. (E) Immunofluorescence analysis was performed to detect the protein expression of CXCR4 (green). The cytoskeleton (F-actin; red) and nuclei (blue) were stained using rhodamine-phalloidin and DAPI, respectively. Scale bars: 50 μm. Data were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests ( n = 3: P < 0.05). Data are presented as the mean ± standard deviation (SD), with different letters indicating statistically significant differences between multiple groups. IL1B, interleukin-1β; MMP8, matrix metalloproteinase 8; TIMP1, tissue inhibitor of matrix metalloproteinases 1; CXCR4, CXC motif receptor type 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDMS, polydimethylsiloxane.

Article Snippet: CXCL12 protein in conditioned medium was measured by Human CXCL12/SDF-1α ELISA Kit (Quantikine, R&D systems.

Techniques: Cell Culture, Microscopy, Quantitative RT-PCR, Gene Expression, Expressing, Control, Immunofluorescence, Staining, Comparison, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Substrate stiffness modulates human gingival fibroblast paracrine signaling to promote osteogenic differentiation of human periodontal ligament cells

doi: 10.3389/fbioe.2026.1753774

Figure Lengend Snippet: Schematic illustration shows the role of human gingival fibroblast–conditioned media (HGF-CM) in regulating osteogenic differentiation of human periodontal ligament cells (HPDLCs). (A) ECM stiffness stimulates CXCL12 chemokine expression in HGFs, which is associated with enhanced osteogenic responses in HPDLCs. (B) A potential clinical application of this mechanism is the utilization of HGF-derived factors to suppress inflammatory bone resorption and stabilize periodontal tissues. CXCL12: CXC motif chemokine 12.

Article Snippet: CXCL12 protein in conditioned medium was measured by Human CXCL12/SDF-1α ELISA Kit (Quantikine, R&D systems.

Techniques: Expressing, Derivative Assay

Journal: Frontiers in Immunology

Article Title: The tropomyosin 3.1/3.2 inhibitor ATM-3507 alters B-cell actin dynamics and impairs the growth and motility of diffuse large B-cell lymphoma cell lines

doi: 10.3389/fimmu.2025.1668379

Figure Lengend Snippet: ATM-3507 inhibits the migration of DLBCL cells toward CXCL12. (A, B) NU-DUL-1 or Toledo cells were pre-treated with ATM-3507 or 0.03% DMSO (equivalent to the highest ATM-3507 concentration) for 1 h before being added to the upper chamber of a Transwell. The lower chamber contained 100 nM CXCL12 plus the same concentration of ATM-3507 or DMSO as in the upper chamber. After 4 h, the number of cells that had migrated into the lower chamber was determined using flow cytometry. The data are expressed as a percent of the number of DMSO-treated cells that migrated into the bottom chamber. The 100% values (percent of DMSO-treated cells that migrated into the lower chamber) in individual experiments ranged from 15-30% for NU-DUL-1 cells and from 50-70% for Toledo cells. Each symbol is an independent experiment. Means ± SEM are shown for 3 independent experiments. (C) NU-DUL-1 or Toledo cells were treated with 3 µM ATM-3507 or 0.03% DMSO for 1 h or 5 h before quantifying cell surface levels of CXCR4 by flow cytometry. Means ± SEM are shown for 3 independent experiments. p-values were calculated using two-tailed paired t -tests.

Article Snippet: Imaging medium (600 μL) with the same concentration of ATM-3507 or DMSO plus 100 nM recombinant human CXCL12 (R&D Systems, Minneapolis, MN, USA #460-SD) was added to the bottom chamber.

Techniques: Migration, Concentration Assay, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: The tropomyosin 3.1/3.2 inhibitor ATM-3507 alters B-cell actin dynamics and impairs the growth and motility of diffuse large B-cell lymphoma cell lines

doi: 10.3389/fimmu.2025.1668379

Figure Lengend Snippet: ATM-3507 inhibits the 2D motility of DLBCL cells on FN. Toledo DLBCL cells were added to FN-coated coverslips in the presence of 100 nM CXCL12 and either 10 µM ATM-3507 or 0.1% DMSO. After a 1 h pre-treatment period, time-lapse images were acquired every 30 s for 1 h. Cell tracks were generated from the time-lapse videos. Representative videos of DMSO-treated cells ( Supplementary Video 1 ) and ATM-3507-treated cells ( Supplementary Video 2 ) are in the Supplementary Material . (A) Individual cell tracks from a representative experiment. In each experiment, 40–100 cell tracks were analyzed per condition. (B) Compiled data from 3 independent experiments. Each symbol represents the median value from an individual experiment. Means ± SEM are shown. p-values were calculated using two-tailed paired t -tests.

Article Snippet: Imaging medium (600 μL) with the same concentration of ATM-3507 or DMSO plus 100 nM recombinant human CXCL12 (R&D Systems, Minneapolis, MN, USA #460-SD) was added to the bottom chamber.

Techniques: Generated, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: The tropomyosin 3.1/3.2 inhibitor ATM-3507 alters B-cell actin dynamics and impairs the growth and motility of diffuse large B-cell lymphoma cell lines

doi: 10.3389/fimmu.2025.1668379

Figure Lengend Snippet: Tpm3.1/3.2 regulates actin organization and actin-dependent processes in B cells. Tpm3.1/3.2 dimers associate with actin filaments, form homopolymers along the filament, stabilize the actin filament, and recruit myosin II. Myosin II dimers can crosslink actin filaments and mediate actomyosin contractility. Inhibition of Tpm3.1/3.2 by ATM-3507 impairs multiple actin-dependent processes in B cells, including the assembly of a peripheral actin ring that drives BCR-induced cell spreading via the formation of protrusive lamellipodia, and the formation of actomyosin arcs (indicated by the yellow arrow) at the inner face of the peripheral actin ring. ATM-3507 also inhibits the growth of DLBCL cells as well as their CXCL12-dependent migration and motility. Created with BioRender.com.

Article Snippet: Imaging medium (600 μL) with the same concentration of ATM-3507 or DMSO plus 100 nM recombinant human CXCL12 (R&D Systems, Minneapolis, MN, USA #460-SD) was added to the bottom chamber.

Techniques: Inhibition, Migration

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A-B) Biolayer interferometry was used to analyze the ligand specificity of Ab93 and ACTRIIA-Fc. At ligand concentrations of 100 nM, associations of rBMP9 and commercial (R&D) rBMP9 were detected for Ab93 (A) , but not other ligands tested (BMP10, activin A, GDF8, GDF11). Under the same conditions, (B) interactions for all other ligands tested, rBMP9, BMP10, activin A, GDF8 and GDF11, were found with ACTRIIA-Fc. (C-D) Circulating levels of BMP9 and BMP10 were measured by a sensitive LC-MS method in plasma obtained from control adult male SD rats, and those undergoing MCT-induced PH for 3 weeks and treated with Ab93 (or isotype control Ab (10 mg/kg i.p. twice weekly) revealing (C) markedly increased levels of circulating BMP9, consistent with trapping and metabolic protection of circulating BMP9, whereas (D) levels of BMP10 were not increased but slightly diminished. Values shown are mean ± S.D., *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001, comparisons based on one-way ANOVA with Sidak’s test. (E-H) Bulk RNAseq analysis of lungs from SU-Hx exposed rats treated with ALK1-Fc versus Ab93 were compared. (E) Heatmap showing DEGs upregulated by SU-Hx in comparison to Normoxic rats and downregulated by ALK1-Fc. (F) H eatmap showing DEGs downregulated by SU-Hx in comparison to Normoxic rats and upregulated by ALK1-Fc. (G) Comparison of numbers of upregulated DEGs in SU-Hx animals that ere downregulated by Ab93 and ALK1-Fc revealed overlapping sets of genes including Cxcl12, Igfbp4, Ccl21, and Grem1 . (H) Genes that were downregulated in SU-Hx rat lungs that were upregulated by treatment of Ab93 or ALK1-Fc included Spn and Rnase6 .

Article Snippet: Recombinant CXCL12, PDGF-BB, TGFβ1, and anti-CXCL12 were purchased from R&D Systems.

Techniques: Liquid Chromatography with Mass Spectroscopy, Clinical Proteomics, Control, Comparison

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Uniform manifold approximation and projection (UMAP) plot showing identified endothelial cell types in lungs from 3 IPAH patients and 6 donor controls (GSE169471). (B) Venn diagram showing the overlap of upregulated genes in SU-Hx rat lung (SU-Hx–Isotype control vs. Nx), downregulated genes in Ab93-treated SU-Hx rat lung (SU-Hx–Ab93 vs. SU-Hx–Isotype), and upregulated genes in IPAH pulmonary general capillary endothelial cells (gCap) (IPAH vs. Control, GSE169471). (C) Venn diagram showing the overlap of upregulated genes in BMP9-treated HPMVEC (BMP9-1.5h vs. control) and upregulated genes in IPAH pulmonary gCap ECs (IPAH vs. Control, GSE169471). (D-G) Volcano plots showing changes and significance of candidate driver genes across different datasets, including (D) upregulation of ID1 , BMPR2 , CXCL12, EDN1, COL18A1 , and IGFBP4 in IPAH vs. control lungs; (E) upregulation of ID1, BMPR2 , EDN1, IGFBP4, VEGFA, PDGFA, PDGFB, VWA1 , and SOX18 in BMP9-treated PMVEC; (F) upregulation of Edn1, Cald1, Vwa1, Col18a1 , and Igfbp4 in Su-Hx treated rats vs. normoxia; and (G) downregulation of Cxcl12, Igfbp4, Col18a1, Vwa1 , and Cald1 in Ab93-treated vs. isotype control treated SU-Hx rats. (H-L) Violin plots showing increased expression of CXCL12 , IGFBP4 , EDN1 , ENG , and BMPR2 in various subsets of endothelial cells from IPAH patients (GSE169471). (M) BMP9 (40 pM) increased CXLC12 mRNA expression in HPMVEC in a time-dependent manner. (N-R) BMP9 (40 pM) increased secretion of CXCL12, IGFBP4, ET-1, PDGF-BB and CCL2 in cultured HPMVEC supernatants. ( n = 3 per group, means ± SD. *P<0.05 as compared to 0 h or all the other groups, Dunnett’s test) (S-U) BMP9 (40 pM) treatment of HPMVEC increased mRNA expression of CXCL12 , which was diminished when BMPR2 , ACVRL1 or ENG were knocked down. ( n = 3 per group, mean ± SD, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 by one-way ANOVA with Sidak’s test).

Article Snippet: Recombinant CXCL12, PDGF-BB, TGFβ1, and anti-CXCL12 were purchased from R&D Systems.

Techniques: Control, Expressing, Cell Culture

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Violin plot showing expression of ACVRL1 in various subpopulations of human pulmonary endothelial cells (GSE169471). (B-D) BMP9 (40 pM) treatment of HPMVEC increased mRNA expression of ENG and BMPR2 , but not ACVRL1 , each of which were diminished by treatment with specific siRNA ( n = 3 per group, mean ± SD, ** P <0.01, *** P <0.001, **** P <0.0001 by one-way ANOVA with Sidak’s test). (E) BMP9 (40 pM) induced expression of CXCL12 mRNA in TIME cells in a manner requiring expression of BMPR2 , ACVRL1 , and ENG based on treatment with specific siRNA. (F) BMP9 (40 pM) induced expression of CXCL12 mRNA in TIME cells in a manner requiring expression of SMAD1 and SMAD5 based on treatment with specific siRNA. Values are shown as mean ± S.D., * P <0.05, ** P <0.01, *** P <0.001, *** P <0.0001, or as indicated by one-way ANOVA with Sidak’s test. (G-M) BrdU incorporation assay showing that CXCL12 inhibited the proliferation of PMVEC from healthy (G) and to a lesser degree PAH donors ( H, I, J) as well as PAEC (K) . CXCL12 and PDGF-BB increased the proliferation of HPASMC ( L and M ). ( n = 8 per group). Values are shown as mean ± S.D., *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or as indicated by t-test.

Article Snippet: Recombinant CXCL12, PDGF-BB, TGFβ1, and anti-CXCL12 were purchased from R&D Systems.

Techniques: Expressing, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Venn diagram of RNA-Seq analysis of whole lung tissues reveals the overlap of upregulated genes in SU-Hx rat lung vs. normoxic rat lungs (SU-Hx up), downregulated genes in Ab93- vs. isotype control treated SU-Hx rat lung (Ab93 down), and upregulated genes in IPAH pulmonary SMC/pericytes (SMC/Pericyte up, GSE169471). (B) Volcano plots depict the expression levels of 6 potentially secreted products of 30 overlapping genes in pulmonary SMC/Pericytes of IPAH patients (GSE169471). (C) Violin plot showing significantly increased Calponin1 (CNN1) expression in pulmonary SMC/pericyte of IPAH patients (GSE169471). (D-H) Treatment with CXCL12 (100 ng/mL) increased expression of contractile phenotype marker genes CNN1 and TAGLN , as well as PAH related genes CPE , THY1 , LTBP2 in monocultured PASMCs. ( n = 4 per group, mean ± SD. ** P <0.01, *** P <0.001, **** P <0.0001 by t-test). (I) PASMCs did not exhibit increased CNN1 expression in response to BMP9 (40 pM) when cultured with other PASMC, but when cocultured with PMVEC exhibited increased CNN1 expression when exposed to BMP9 ( n = 3 to 4 per group, mean ± SD, ** P <0.01 by t-test. (J-K) Treatment of co-cultured PMVEC and PASMC with BMP9 induced CNN1 expression in PASMC. BMP9 treatment-induced expression of CNN1 in PASMC cocultured with PMVEC (J) , which was partially blocked by CXCL12 neutralizing antibody (5 nM, n = 3 per group, mean ± SD. * P <0.05, ** P <0.01, **** P <0.0001 by one-way ANOVA with Holm-Sidak test). (L) Treatment of co-cultured PMVEC and PASMC with 5% human serum induced CNN1 expression in HPASMCs, which was partially neutralized by treatment with anti-BMP9 (MAB3209, 5 mM, n = 3 per group, ** P <0.01, **** P <0.0001 by one- way ANOVA with Dunnett’s test). (M) BMP9 treatment-induced expression of CNN1 in PASMC that were co-cultured with PMVEC was inhibited by treatment with CXCR4 inhibitor AMD3100 (10 µM, n = 4 per group, mean ± SD. ** P <0.01, *** P <0.001 by one-way ANOVA with Dunnett’s test).

Article Snippet: Recombinant CXCL12, PDGF-BB, TGFβ1, and anti-CXCL12 were purchased from R&D Systems.

Techniques: RNA Sequencing, Control, Expressing, Marker, Cell Culture

Journal: Heliyon

Article Title: Diphlorethohydroxycarmalol inhibits Müller cell gliosis by disrupting CXCR4/CXCL12 interaction in violet-blue light-induced retinal phototoxicity.

doi: 10.1016/j.heliyon.2025.e42475

Figure Lengend Snippet: Fig. 1. In vitro modeling of the effect of BL exposure on CXCL12 and CXCR4 expression (A) Schematic image showing the establishment of an in vitro model of BL damage using a BL LED device. Figure (A) generated with BioRender (https://biorender.com/). (B) After a 100 min of BL exposure, a cytokine array analysis was conducted. The protein expression of chemokine (C-C motif) ligand 5 (also known as CCL5) (indicated by the black box) decreased in the BL group compared to the dark group, but there were no changes in the BL/DPHC groups. (B, C) Conversely, CXCL12 protein expression (indicated by the red box) increased only in the MIO-M1 cell group exposed to BL (BL group) compared to the MIO-M1 cells exposed to darkness (Dark group). However, the CXCL12 protein expression decreased in the DPHC-treated BL-exposed MIO-M1 cell group (the BL/ DPHC groups) compared to the BL group. (D–F) BL exposure increased (D, E) the protein expression of CXCR4, which is the receptor of CXCL12, compared to the dark group. Interestingly, (F) the CXCR4 mRNA expression followed a similar pattern to the protein expression. DPHC treatment decreased both the protein and mRNA expression of CXCR4. The results presented in this study are expressed as the mean ± SEM. One-way ANOVA was conducted, followed by Tukey’s multiple comparisons test (p < 0.05). Distinct letters (a, b, c, d) above the bars denote groups that differ significantly (p < 0.05), while bars sharing the same letter are not significantly different. Each experiment was performed at least three times independently. Scale bar = 100 μm, BL, blue light; CCL5, chemokine (C-C motif) ligand 5; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4; DPHC, diphlorethohydroxycarmalol.

Article Snippet: In another in vitro model, CXCL12 induction was achieved by treating MIO-M1 cells with 20 nM recombinant CXCL12 protein (R&D Systems) for 1 d. Prior to the induction of CXCL12 in MIO-M1 cells, DPHC was pre-treated with the cells for 1 d.

Techniques: In Vitro, Expressing, Generated

Journal: Heliyon

Article Title: Diphlorethohydroxycarmalol inhibits Müller cell gliosis by disrupting CXCR4/CXCL12 interaction in violet-blue light-induced retinal phototoxicity.

doi: 10.1016/j.heliyon.2025.e42475

Figure Lengend Snippet: Fig. 3. Validation of reactive Müller glia markers in DPHC-treated MIO-M1 cells after BL damage or CXCL12 induction (A) To validate the reactive Müller glia marker expression in MIO-M1 cells, GFAP and GS protein expression was characterized using western blot analysis after BL exposure. Standard protein markers were loaded in the far left lane. The uncropped and non-adjusted images can be found in the supplementary Fig. 2 (B, C) The graphs show the expression intensity of GFAP and GS after BL damage. (D) The cell images demonstrate changes in cell morphology after BL damage. Scale bar = 200 μm (E) BL damage altered the soma area or cell body. (F) Schematic image showing the establishment of an in vitro model of CXCL12 induction. Figure (F) generated with BioRender (https://biorender.com/). (G–I) To validate the reactive Müller glia marker expression in CXCL12 induced MIO-M1 cells, GFAP and GS protein expression was characterized using immunocytochemistry. Scale bar = 100 μm (H, I) The quantified graphs show GFAP and GS expression levels after CXCL12 induction. The results presented in this study are expressed as the mean ± SEM. One-way ANOVA was conducted, followed by Tukey’s multiple comparisons test (p < 0.05). Distinct letters (a, b, c, d) above the bars denote groups that differ significantly (p < 0.05), while bars sharing the same letter are not significantly different. Each experiment was performed at least three times independently. Scale bar = 100 μm, BL, blue light; DPHC, diphlorethohydroxycarmalol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase.

Article Snippet: In another in vitro model, CXCL12 induction was achieved by treating MIO-M1 cells with 20 nM recombinant CXCL12 protein (R&D Systems) for 1 d. Prior to the induction of CXCL12 in MIO-M1 cells, DPHC was pre-treated with the cells for 1 d.

Techniques: Biomarker Discovery, Marker, Expressing, Western Blot, In Vitro, Generated, Immunocytochemistry

Journal: Heliyon

Article Title: Diphlorethohydroxycarmalol inhibits Müller cell gliosis by disrupting CXCR4/CXCL12 interaction in violet-blue light-induced retinal phototoxicity.

doi: 10.1016/j.heliyon.2025.e42475

Figure Lengend Snippet: Fig. 4. Validation of CXCR4-NF-κB signaling in DPHC-treated MIO-M1 cells after BL damage (A) Schematic depiction of the role of CXCR4-NF- κB signaling in glial gliosis. In DPHC-treated MIO-M1 cells, DPHC tends to bind to CXCR4 instead of its ligand CXCL12. Figure (A) generated with BioRender (https://biorender.com/). (B–D) (B) The results of western blot analysis and protein expression quantification revealed that DPHC treatment resulted in altered phosphorylation of (C) ERK1/2 (Thr183/Tyr 185), (D) AKT1/2/3 (Thr 308/Ser 473). The uncropped and non-adjusted images can be found in the Supplementary Fig. 3. (E) The translocation of pNF-κB from the cytoplasm to the nucleus was validated using immu nocytochemistry. Arrows indicate expression of pNF-κB in nucleus of MIO-M1 cells. Scale bar = 50 μm Standard protein markers were loaded in the far left lane. The results presented in this study are expressed as the mean ± SEM. One-way ANOVA was conducted, followed by Tukey’s multiple comparisons test (p < 0.05). Distinct letters (a, b, c, d) above the bars denote groups that differ significantly (p < 0.05), while bars sharing the same letter are not significantly different. Each experiment was performed at least three times independently. AKT1/2/3, Akt serine/threonine kinase family 1/2/3; BL, blue light; DPHC, diphlorethohydroxycarmalol; ERK1/2, extracellular signal-regulated kinase 1/2; GAPDH, glyceraldehyde 3- phosphate dehydrogenase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.

Article Snippet: In another in vitro model, CXCL12 induction was achieved by treating MIO-M1 cells with 20 nM recombinant CXCL12 protein (R&D Systems) for 1 d. Prior to the induction of CXCL12 in MIO-M1 cells, DPHC was pre-treated with the cells for 1 d.

Techniques: Biomarker Discovery, Generated, Western Blot, Expressing, Phospho-proteomics, Translocation Assay

Journal: Heliyon

Article Title: Diphlorethohydroxycarmalol inhibits Müller cell gliosis by disrupting CXCR4/CXCL12 interaction in violet-blue light-induced retinal phototoxicity.

doi: 10.1016/j.heliyon.2025.e42475

Figure Lengend Snippet: Fig. 5. Inhibitory effects of DPHC on Müller gliosis of zebrafish eyes after BL damage (A) Schematic illustrating the role of DPHC in the development of gliosis in zebrafish. To induce retinal gliosis, zebrafish were exposed to BL for 4 h per day over a 3 d period. Figure (A) generated with BioRender (https://biorender.com/). (B, C) Histological changes in the zebrafish retina were assessed through H&E staining, and the thickness of the retina was measured using the ImageJ software. (D) The red arrowheads indicate CXCL12 expression in the cells, whereas the yellow ar rowheads indicate the absence of CXCL12 staining. (E, F) TUNEL staining, which detects apoptotic retinal cells, was used to stain the positive cells (white), whereas DAPI stained the nucleus in blue. The TUNEL-positive cells (white) were counted using the ImageJ software and the graph represents the quantification of apoptotic cell numbers derived from representative images. The results presented in this study are expressed as the mean ± SEM. One-way ANOVA was conducted, followed by Tukey’s multiple comparisons test (p < 0.05). One-way ANOVA was conducted, fol lowed by Tukey’s multiple comparisons test (p < 0.05). Distinct letters (a, b, c, d) above the bars denote groups that differ significantly (p < 0.05), while bars sharing the same letter are not significantly different. Each experiment was performed at least three times independently. Scale bar = 20 μm, BL, blue light; CXCL12, C-X-C motif chemokine ligand 12; DAPI, 4′,6-diamidino-2-phenylindole; DPHC, diphlorethohydroxycarmalol; H&E, hematoxylin & eosin, INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PRL, photoreceptor layer; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: In another in vitro model, CXCL12 induction was achieved by treating MIO-M1 cells with 20 nM recombinant CXCL12 protein (R&D Systems) for 1 d. Prior to the induction of CXCL12 in MIO-M1 cells, DPHC was pre-treated with the cells for 1 d.

Techniques: Generated, Staining, Software, Expressing, TUNEL Assay, Derivative Assay